New antibiotics based on novel chemical scaffolds are vital to the biodefense armory primarily because they will be effective against both natural and engineered resistant forms of bioterrorist microbes. In addition, the absence of a reservoir of pre-existing resistance alleles may prolong their useful lifetime. The goal of this research is to discover and develop novel antibiotics effective against B. anthracis, including resistant forms of this organism, for biodefense. Our strategy is to screen for novel inhibitors of the DNA replication pathway, which consists of multiple essential, drug-validated targets. In Phase I, we will engineer the non-pathogenic B. anthracis Sterne strain to build a permeable-cell DNA replication pathway screen. We will validate this screen, optimize it for high throughput, and apply it to a diverse library of over 100,000 discrete chemical and natural product structures. Compounds that inhibit DNA replication will be confirmed in replicate assays and evaluated in secondary screens against two essential DNA replication targets -- DNA polymerase III C (pol lII C) and topoisomerase IV (topo IV). These enzymes will be prepared through the cloning and expression of genes isolated from B. anthracis genomic DNA. In addition, we will apply these secondary assays to focused compound libraries built around three chemical cores known to inhibit pol IIIC and topo IV in pathogenic, but non-bioterrorist Gram(+) bacteria in order to identify suitable derivatives for optimization as B. anthracis inhibitors. Finally, we will test the resulting validated hits for purity and mass, potency on intact B. anthracis Sterne cells, specificity for inhibition of DNA replication, selectivity with respect to mammalian cells in culture, and other drug-like properties.
The specific aims are to (1) develop a permeable-cell, high-throughput DNA replication pathway assay using B. anthracis Sterne; (2) construct biochemical secondary assays for pol III C and topo IV from B. anthracis; (3) screen a diverse compound library with the high-throughput DNA replication assay and confirm resulting hits; and (4) profile confirmed hits for activity on bacterial cells, specificity for DNA synthesis inhibition, and mammalian cell cytotoxicity. In Phase II, we will optimize the most promising compounds to develop novel leads and candidate drugs for pre-clinical testing in animal models of infection. ? ?
